Nitric oxide (NO) is a toxic gas encountered by bacteria as a product of their own metabolism or as a result of a host immune response. Non-toxic concentrations of NO have been shown to initiate changes in bacterial behaviors such as the transition between planktonic and biofilm-associated lifestyles. The heme nitric oxide/oxygen binding proteins (H-NOX) are a widespread family of bacterial heme-based NO sensors that regulate biofilm formation in response to NO. The presence of H-NOX in several human pathogens combined with the importance of planktonic-biofilm transitions to virulence suggests that H-NOX sensing may be an important virulence factor in these organisms. Here we review the recent data on H-NOX NO signaling pathways with an emphasis on H-NOX homologues from pathogens and commensal organisms. The current state of the field is somewhat ambiguous regarding the role of H-NOX in pathogenesis. However, it is clear that H-NOX regulates biofilm in response to environmental factors and may promote persistence in the environments that serve as reservoirs for these pathogens. Finally, the evidence that large subgroups of H-NOX proteins may sense environmental signals besides NO is discussed within the context of a phylogenetic analysis of this large and diverse family.

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Bacterial Heme‐Nitric Oxide/Oxygen binding (H‐NOX) proteins utilize heme to act as diatomic gas sensors. These proteins often interact with a signaling partner to govern communal behaviors such as quorum sensing and biofilm formation in response to nitric oxide in facultative anaerobes. H‐NOX from Vibrio cholerae (Vc H‐NOX) in the Fe(III) and Fe(II)‐NO states was demonstrated to inhibit the activity of a cognate histidine kinase (HK). Heme‐free Vc H‐NOX could also act as an inhibitor of HK signaling through reversible oxidation of cysteine residues at a zinc binding site, suggesting that H‐NOX proteins may act as redox sensors in some organisms. The zinc‐binding Cys residues are conserved across numerous species, including the aquatic organism and obligate aerobe, Caulobacter crescentus (Cc H‐NOX). Using UV‐Vis spectroscopy, our lab has characterized heme complexes from Cc H‐NOX as purified, reduced, CO‐bound, and NO‐bound. ICP‐OES has provided insight to the degree of heme binding in Cc H‐NOX samples as well as zinc content. Circular dichroism has provided an estimate of secondary structure and a melt curve using single wavelength readings with variable temperatures. Lastly, we have generated a knockout mutant to investigate the importance of H‐NOX in biofilm regulation and the impact this knockout has on the Caulobacter crescentus proteome. Our results have shown complete zinc binding for Cc H‐NOX, the formation of a stable NO‐complex, and a mild hyper‐biofilm phenotype among Caulobacter crescentus mutants. The mutant also exhibits differences in protein expression levels and phosphorylation reflecting signaling pathways altered in the absence of H‐NOX. This has led to an improved understanding of the roles of H‐NOX signaling across bacterial species.